Identification of the parameters that had significant effects on  polyunsaturated fatty acids (PUFAs) and biomass production by the moss Physcomitrella patens was performed using nine culture variables (temperature, agitation speed, pH, sucrose, di-ammonium tartrate, CaCl₂.2H₂O, MgSO₄.7H₂O, KH₂PO₄ and KNO₃) with the statistical design technique of Plackett-Burman. Statistical analysis revealed that two physical variables (pH and temperature) had significant effects on the production of both biomass and PUFAs (linoleic acid, LA; g-linolenic acid, GLA; a-linolenic acid, ALA; eicosadienoic acid, EDA; di-homo-g-linolenic acid, DHGLA; arachidonic acid, ARA; eicosapentaenoic acid, EPA). Three nutritional variables (sucrose, CaCl₂ and MgSO₄) had an influence only on the production of some of the PUFAs. Of the two levels used in this study, higher concentrations of sucrose had a positive effect on LA, ARA and EPA production, whereas higher concentrations of metal ions (CaCl₂ and MgSO₄) had a negative effect only on ARA and EPA production.
Adrenic acid (ADA), an w-6 PUFA) naturally absent in P. patens, has attracted much interest due to its pharmaceutical potential. Exploiting the wealth of information currently available on in planta oil biosynthesis, and coupling this information with the tool of genetic engineering, it is now feasible to deliberately alter fatty acid biosynthetic pathways to generate unique oils in commodity crops. In this study, a D⁵-elongase gene from the algae Pavlova sp. related to the biosynthesis of C₂₂-PUFAs was targeted to enable production of ADA in P. patens. Heterologous expression of this gene was under the control of a tandemly duplicate 35S promoter. It was established that ADA (0.42 mg/l) was synthesized in P. patens from endogenous ARA via the expressed Pavlova sp. D⁵-elongase in the moss. In an attempt to maximize ADA production, medium optimization was effected by the response surface methodology (RSM), resulting in a significant elevation of ADA (4.51 mg/l) production under optimum conditions. To the best of our knowledge, this is the first study describing the expression of a PUFA synthesizing enzyme in non-seed lower plant without supplying the exogenous fatty acid.